Intestinal intraepithelial lymphocytes prevent pathogen-driven inflammation and regulate the Smad/T-bet pathway of lamina propria CD4+ T cells

Intraepithelial lymphocytes (IEL) play a key role in gut homeostasis and are critical effector cells preventing the inflammatory intestinal lesions induced in mice following oral infection with Toxoplasma gondii. In this intestinal inflammatory model, CD4(+) T lymphocytes from the lamina propria (LP) synergize with the infected enterocytes to secrete pro-inflammatory chemokines and cytokines. In this study, we assessed the mechanisms accounting for the ability of IEL to modulate the inflammatory activity of these cells. Adoptive transfer of IEL purified from wild-type mice, or CD154-,CD95L- or IL-10-deficient mice infected with T. gondii completely impairs the development of the lethal ileitis in recipient mice orally infected with T. gondii.Compared with unprimed IEL isolated from naive mice, the CD8 alpha beta TCR alpha beta subset of primed IEL, isolated from T. gondii-infected mice, secretes increased amount of TGF-beta. IEL interact with the LP CD4(+) T lymphocytes, down-regulate their production of inflammatory cytokines such as IFN-gamma and reduce their proliferative activity. These effects are linked to the secretion of TGF-beta and are correlated with a shift in the balance between Smad7/T-bet down-regulation and Smad2/Smad3 up-regulation in LP CD4(+) T lymphocytes.     

Effect of specific antigen stimulation on intraepithelial lymphocyte migration to small intestinal mucosa

Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.     

Intestinal intraepithelial lymphocytes sustain the epithelial barrier function against Eimeria vermiformis infection

Eimeria spp. are intracellular protozoa that infect intestinal epithelia of most vertebrates, causing coccidiosis. Intestinal intraepithelial lymphocytes (IEL) that reside at the basolateral site of epithelial cells (EC) have immunoregulatory and immunoprotective roles against Eimeria spp. infection. However, it remains unknown how IEL are involved in the regulation of epithelial barrier during Eimeria sp. infection. Here, we demonstrated two distinct roles of IEL against infection with Eimeria vermiformis, a murine pathogen: production of cytokines to induce protective immunity and expression of junctional molecules to preserve epithelial barrier. The number of IEL markedly increased when oocyst production reached a peak. During infection, IEL increased production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and decreased transforming growth factor beta (TGF-beta) production. Addition of IFN-gamma and TNF-alpha or supernatants obtained from cultured IEL from E. vermiformis-infected mice reduced transepithelial electrical resistance (TER) in a confluent CMT93 cell monolayer, a murine intestine-derived epithelial line, but antibodies against these cytokines suppressed the decline of TER. Moreover, TGF-beta attenuated the damage of epithelial monolayer and changes in TER caused by IFN-gamma and TNF-alpha. The expression of junctional molecules by EC was decreased when IEL produced a high level of IFN-gamma and TNF-alpha and a low level of TGF-beta in E. vermiformis-infected mice. Interestingly, IEL constantly expressed junctional molecules and a coculture of EC with IEL increased TER. These results suggest that IEL play important multifunctional roles not only in protection of the epithelium against E. vermiformis-induced change by cytokine production but also in direct interaction with the epithelial barrier when intra-EC junctions are down-regulated.     

Transforming growth factor-beta mediates intestinal healing and susceptibility to injury in vitro and in vivo through epithelial cells

In vitro studies suggest that transforming growth factor (TGF)-beta has potent effects on gastrointestinal mucosal integrity, wound repair, and neoplasia. However, the multiplicity of actions of this peptide on many different cell types confounds efforts to define the role of TGF-beta within the intestinal epithelium in vivo. To delineate these effects selective blockade of intestinal epithelial TGF-beta activity was undertaken through targeted expression of a dominant-negative (DN) TGF-beta RII to intestinal epithelial cells in vitro and in vivo. Stable intestinal epithelial cell (IEC)-6 lines overexpressing TGF-beta RII-DN (nucleotides -7 to 573) were established. Transgenic mice overexpressing TGF-beta RII-DN under the regulation of a modified liver fatty acid-binding promoter (LFABP-PTS4) were constructed. In vitro healing was assessed by wounding of confluent monolayers. Colitis was induced by the addition of dextran sodium sulfate (2.5 to 7.5% w/v) to their drinking water. Overexpression of TGF-beta RII-DN in intestinal epithelial cell-6 cells resulted in a marked reduction in cell migration and TGF-beta-stimulated wound healing in vitro. TGF-beta RII-DN transgenic mice did not exhibit baseline intestinal inflammation or changes in survival, body weight, epithelial cell proliferation, aberrant crypt foci, or tumor formation. TGF-beta RII-DN mice were markedly more susceptible to dextran sodium sulfate-induced colitis and exhibited impaired recovery after colonic injury. TGF-beta is required for intestinal mucosal healing and TGF-beta modulation of the intestinal epithelium plays a central role in determining susceptibility to injury.     

Influence of enterotoxin on mucosal intranet: selective inhibition of extrathymic T cell development in intestinal intraepithelial lymphocytes by oral exposure to heat-labile toxin

We tested the possibility that heat-labile enterotoxin of Escherichia coli (LT) affects the development of extrathymic T cells in the intraepithelial lymphocyte (IEL) compartment. After oral administration of LT, the number of extrathymic CD8alphaalpha+ IEL was selectively and significantly diminished when compared with the corresponding cells in phosphate-buffered saline-fed control mice. To clarify the mechanism behind this selective reduction of CD8alphaalpha+ IEL, we analyzed the expression of essential cytokines and their corresponding receptors for the mucosal intranet formed by intestinal epithelial cells (IEC) and IEL. The expression levels of stem cell factor, interleukin (IL)-7, and IL-15 in IEC, and their corresponding receptors, i. e. c-kit, IL-7 receptor, and IL-15 receptor, in CD8alphaalpha+ IEL were reduced following oral feeding with LT. These findings suggest that LT negatively regulates development of CD8alphaalpha+ IEL via the disruption of mucosal intranet-associated cytokine and cytokine receptors, which are required for the development and/or expansion of extrathymically developed T cells. Further, LT-induced destruction of the mucosal intranet resulted in the impairment of IEC generation via an increase of apoptosis.     

Inhibitory effects of transforming growth factor-beta (TGF-beta) on certain functions of intraepithelial lymphocytes

Human intraepithelial lymphocytes (IEL), CD8+ lymphocytes located between epithelial cells, are likely to be influenced by the immunosuppressive cytokine, TGF-beta, secreted by epithelial cells. This study evaluates the effects of TGF-beta on IEL functions. IEL were derived from proximal jejunum of patients undergoing gastric bypass operations for morbid obesity. Proliferation was determined by 3H-thymidine incorporation; IL-2 production, by ELISA; expression of IL-2 receptor, CD2, HML1, CD16, and CD56, by immunofluorescence; binding, by adherence of radiolabelled cells; and cytotoxicity by 51Cr-release assay. TGF-beta (> or = 1 ng/ml) inhibited the mitosis of IEL to mitogens, IL-7, and stimuli of the CD2 and CD3 pathways. The blocking effect did not target the activation events of IL-2 production and receptor generation. Rather, it reduced cell division after activation when added 24 h after initiating the culture. Antibody neutralization of naturally occurring TGF-beta increased IEL proliferation to IL-2, but not to the other stimuli. Of the multiple surface markers tested, only CD2 and HML1 expression increased with TGF-beta and decreased with antibody to TGF-beta, although the cytokine and the neutralizing antibody had no effects on IEL binding to colon cancer. TGF-beta reduced the number of CD56+ IEL and the lymphokine-activated killing when co-cultured with IL-7 but not with IL-2 or IL-15. TGF-beta inhibits certain IEL functions: the reduction in cell division rather than activation and a decline in IL-7-mediated lysis of colon cancer due to a lowering of the number of natural killer cells.     

Hepatocyte growth factor inhibits epithelial to myofibroblast transition in lung cells via Smad7

Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-beta-induced expression of myofibroblast markers such as alpha-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-beta-induced alpha-SMA expression in primary murine alveolar epithelial cells. Since TGF-beta is known to regulate alpha-SMA expression, the effect of HGF on components of TGF-beta signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-beta signaling, in a mitogen-activated protein kinase-dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in alpha-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.     

Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury during murine giardiasis

T-cell-mediated pathogenesis has been documented in various idiopathic and microbially induced intestinal disorders. Diffuse microvillous shortening seen in giardiasis is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water. Other mucosal changes include crypt hyperplasia and increased numbers of intraepithelial lymphocytes (IEL). A recent report using an athymic mouse model of infection showed that these epithelial injuries were dependent on T cells. The aim of the present study was to identify which subset of superior mesenteric lymph node (SMLN) T cells were responsible for mucosal alterations in giardiasis. CD4+ and CD8+ T cells, as well as whole lymphocyte populations, were isolated from SMLN of Giardia muris-infected mice for adoptive transfer. Jejunal segments of recipient mice were assessed for brush border ultrastructure, sucrase activity, crypt/villus ratio, and IEL numbers. Mice that received enriched CD8+ and whole SMLN lymphocytes, but not CD4+ T cells, from infected donors showed diffuse shortening of microvilli, loss of brush border surface area, impaired sucrase activity, and increased crypt/villus ratios compared to respective controls. Transfer of whole SMLN lymphocytes, as well as enriched CD4+ or CD8+ T cells, from infected donors led to increased IEL numbers in the recipient jejunum. The findings indicate that loss of intestinal brush border surface area, reduced disaccharidase activities, and increased crypt/villus ratios in giardiasis are mediated by CD8+ T cells, whereas both CD8+ and CD4+ SMLN T cells regulate the influx of IEL.     

Smad7 in TGF-beta-mediated negative regulation of gut inflammation

Mice with targeted disruptions of the transforming growth factor-beta1 (TGF-beta1) gene or TGF-beta1 intracellular signalling pathways develop intestinal inflammation. Conversely, TGF-beta1-producing regulatory T cells protect against experimental colitis. Paradoxically, however, TGF-beta1 production is high in the gut of patients with chronic inflammatory intestinal disease, and yet inflammation proceeds unchecked. Here we discuss the functional role of Smad7, an intracellular inhibitor of TGF-beta1 signalling, in the control of gut inflammation by TGF-beta1. In particular, we delineate a scenario in which the high expression of Smad7 in inflammatory cells renders them unresponsive to TGF-beta1 and propose that control of Smad7, not TGF-beta1 production, is a key determinant in understanding how TGF-beta1 negatively regulates gut inflammation.     

Differential expression of CD43 isoforms on murine T cells and their relationship to acute intestinal graft versus host disease: studies using enhanced-green fluorescent protein transgenic mice.

Three mAb (R2/60, S7 and 1B11) were used to study the expression of murine CD43 on peripheral T cells and intestinal intraepithelial lymphocytes (IEL) from normal mice, and from mice during acute graft versus host disease (GVHD). In the spleen, essentially all T cells expressed the R2/60 and S7 antigens, whereas the 1B11 antigen was expressed on about half of the CD8(+) cells and approximately 15% of CD4(+) T cells. Interestingly, a significant proportion of resting splenic B cells expressed the 1B11 and R2/60 antigens, but not the S7 antigen. The majority of IEL expressed R2/60 antigen; however, the S7 and 1B11 markers were differentially expressed on CD8alpha, CD8beta, TCRalphabeta and TCRgammadelta cells. Immunoprecipitation and Western blotting analyses identified characteristic 115 and 130 kDa reactive components from IEL lysates with mAb S7 and 1B11 respectively, and reactivity to both molecular entities by mAb R2/60. During acute intestinal GVHD induced by injecting CB6F(1) athymic nude mice with spleen cells from C57BL/6 enhanced-green fluorescent protein transgenic mice, 80-90% of donor T cells in the intestine epithelium expressed all CD43 isoforms; however, the level of expression of the 130 kDa CD43 antigen increased significantly and the level of the 115 kDa antigen decreased on GVHD donor T cells compared to cells at the time of transfer. Using EL4 cells, a similar shift in the expression of CD43 isoforms occurred experimentally following treatment with neuraminidase, suggesting that the type of CD43 isoform expressed on T cells is strongly influenced by conditions which affect membrane charge. The significance of these findings for intestinal immunopathology is discussed.     

Expression of mucosa-related integrin alphaEbeta7 on alveolar T cells in interstitial lung diseases.

The expression of alphaEbeta7 integrin has been related to the selective retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. To identify potential disease-associated alphaEbeta7 expression patterns on cells accounting for lymphocytic alveolitis in interstitial lung disease (ILD), alphaE expression on CD4+ and CD8+ T cell subsets was evaluated by dual-colour flow cytometry in peripheral blood and bronchoalveolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF; n = 18), hypersensitivity pneumonitis (HP; n = 20) and sarcoidosis (n = 44) in comparison with healthy controls (n = 15). In both healthy individuals and all patient groups the proportion of alphaE-bearing T cells in peripheral blood was < 2%, whereas the vast majority of alveolar CD8+ T cells consistently co-expressed alphaE. Absolute alveolar CD8+alphaE+ cell numbers/ml were up to 30-fold increased in HP patients. Proportions of alphaE-bearing CD4+ cells in BALF were significantly elevated in IPF (74.0 +/- 2.7%) and HP (70.0 +/- 2.4%) compared with normals (30.0 +/- 1.8%) (mean +/- s.e.m.; P < 0.01). In sarcoidosis, the alphaE expression on BALF CD4+ cells displayed subgroup dependency: proportions significantly lower than normal were noted in chest radiographic stage I (14.3 +/- 1.5%), but increased proportions in stages II (50.0 +/- 3.8%) and III (64.0 +/- 4.8%). Correlations between common markers of T cell activation or BALF transforming growth factor-beta (TGF-beta ) bioactivity and alphaE expression were not noted. We conclude that the vast majority of alveolar CD8+ T cells consistently express alphaEbeta7 and that distinct patterns of alphaEbeta7 expression on alveolar CD4+ lymphocytes in sarcoidosis are related to the diverse manifestations of the sarcoid inflammatory process in the lung.     

Localization and phenotype of CD3 associated gamma/delta receptor expressing intestinal intraepithelial lymphocytes

This study was carried out to determine the exact phenotype and localization of intestinal intra epithelial lymphocytes (IEL). IEL reside in the small intestine between or just beneath the epithelial cell layer which covers the lamina propria. These CD3 positive cells were found in equal numbers at this location both in the villi as well as in the crypts. The majority of these cells express a CD3 associated gamma delta receptor. Cell surface marker analysis of isolated IEL reveals high percentages of cells expressing CD8 and/or Thy-1. No CD4 positive cells could be detected in significant numbers. Equal quantities of IEL could be isolated from the intestine of athymic (nude) mice. Although the percentage of CD3 positive cells in the IEL from nude mice was lower than the figures obtained from euthymic mice northern blot analysis revealed a strong expression of gamma delta message in the IEL of nude mice. In the CD3 positive IEL fraction of nude mice the relative contribution of the CD8 positive cells remained unchanged, whereas the percentage of Thy-1 positive cells had decreased. Still significant numbers of such cells could be demonstrated in the IEL of nude mice. Altogether these results show that, at least, a significant part of the gamma delta receptor bearing IEL is independent of the thymus for their differentiation.     

Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation

To determine which chemokine receptors might be involved in T lymphocyte localization to the intestinal mucosa, we examined receptor expression on human intestinal lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL) and CD45RO+beta7hi gut homing peripheral blood lymphocytes (PBL). Virtually all LPL and IEL expressed CXCR3 and CCR5, receptors that have been associated with Th1(Tc1)/Th0 lymphocytes, while CCR3 and CCR4, receptors associated with Th2 (Tc2)lymphocytes, CCR7, CXCR1 and CXCR2 were not expressed. CXCR3 and CCR5 receptors were functional, as LPL and IEL migrated to their respective ligands I-TAC and RANTES. In addition, most alphaEbeta7- LPL and IEL expressed high levels of CCR2. While the majority of CD45RO(-)beta7hi PBL also expressed CXCR3 and CCR5, a proportion of these cells were CXCR3- and/or CCR5- and some expressed CCR4 and/or CCR7, indicating that lymphocytes recruited to the intestinal mucosa represent a subset of these cells. In summary, our results show that LPL and IEL within the normal intestine express a specific and similar array of chemokine receptors whose ligands are constitutively expressed in the intestinal mucosa and whose expression is up-regulated during intestinal inflammation. These results support the view that CXCR3, CCR5 and CCR2 may play an important role in lymphocyte localization within the intestinal mucosa.     

Presence of intestinal intraepithelial lymphocytes in mice with severe combined immunodeficiency disease.

The murine intestinal epithelium contains a heterogeneous population of intraepithelial leukocytes (IEL) most of which are granulated, Thy-1-CD5-CD8+. In order to assess the lineage relationship of this subgroup of IEL to peripheral T cells, we examined IEL in mice with the severe combined immunodeficiency (scid/scid) mutation, which lack T and B cells in peripheral lymphoid tissues. Electron and light microscopy showed that the intestine from scid/scid mice had granulated IEL similar to IEL in normal C.B-17 mice. Flow cytometry of isolated IEL stained with monoclonal antibodies against Thy-1, CD3, CD4, CD5 and CD8 showed that scid/scid mice IEL contained cells with the Thy-1-CD4-CD5-CD8+ phenotype. Immunohistochemical staining of IEL in tissue sections with antibodies to Thy-1 and CD8 confirmed that the Thy-1-CD8+ cells were in the intestinal epithelium. These scid/scid IEL also lacked CD3 expression and mRNA for the V gamma 7 V region gene of the gamma T cell receptor. We conclude that scid/scid mice contain precursors for IEL that can differentiate into a granulated Thy-1-CD5-CD8+ IEL in the intestine. The absence of CD8+ peripheral T cells in these mice suggests that these IEL differ from classical T cells in their ability to differentiate and express CD8 and do not require T cell receptor expression for their localization to the intestine.     

Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.     

Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali

Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.     

The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes

Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.